WO2018113788A1 - 重组单链人FVIII-Fc融合蛋白及其应用 - Google Patents
重组单链人FVIII-Fc融合蛋白及其应用 Download PDFInfo
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- fviii
- fusion protein
- recombinant single
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/37—Factors VIII
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the present application relates to the field of recombinant FVIII-Fc fusion proteins, and more particularly to fusion proteins formed by the association of a B domain deleted human FVIII with a particular single chain Fc.
- Hemophilia A is an inherited bleeding disorder caused by a deficiency or dysfunction of factor VIII (FVIII) activity. Supplementation with active FVIII is an effective measure for the treatment of hemophilia A.
- FVIII factor VIII
- the circulating half-life of endogenous FVIII is 12-14 hours, so several prophylactic treatments are needed each week. In order to improve patient compliance and reduce the number of weekly FVIII administrations, researchers have been working to develop FVIII with extended half-life.
- FVIII has a large molecular weight and is the most active protein expressed by the current recombinant technology, and it needs to form a complex with phospholipids, FIXa and FX to exert clotting activity. Therefore, the bivalent molecular form of FVIII-Fc protein will be sterically hindered. The effect results in a decrease or loss of FVIII activity.
- Patent CN201080062950.7 discloses a FVIII-Fc chimeric protein, wherein the chimeric protein comprises two strands, one strand comprising FVIII and the hinge region of IgG1 -CH2-CH3, and the other strand comprising only the hinge region of IgG1 -CH2 -CH3, the two chains are dimerized by the Fc region to form a hybrid protein.
- the chimeric FVIII-Fc yield is extremely low due to the simultaneous production of a "hinge region-CH2-CH3" homodimeric form of the molecule, and cannot be purified by conventional protein A.
- the process of obtaining the protein of interest can only be purified by VIIIselect (GE Healthcare), which greatly increases the production cost.
- the present application aims to provide a novel recombinant single-stranded human FVIII-Fc fusion protein in which two CH2-CH3 domains in an Fc fragment are joined into a single strand by a flexible polypeptide. Fusion protein expression was performed using a eukaryotic cell expression system. The expressed recombinant single-stranded human FVIII-Fc can be obtained by a conventional protein A purification process to obtain a higher purity protein.
- the application first provides a recombinant single-stranded human FVIII-Fc fusion protein, wherein FVIII is a B domain deleted human FVIII; Fc is derived from human IgG1 comprising a N297G mutation, and two CH2-CH3 domains are joined by a flexible polypeptide.
- FVIII is a B domain deleted human FVIII
- Fc is derived from human IgG1 comprising a N297G mutation, and two CH2-CH3 domains are joined by a flexible polypeptide.
- a single strand introduced T366S, L368A and Y407V mutations in CH3 near the N-terminus and a T366W mutation in CH3 near the C-terminus.
- the FVIII is linked directly to the Fc or via a flexible polypeptide.
- the flexible polypeptide consists of at least one amino acid selected from the group consisting of G, S, A, T.
- the recombinant single-stranded human FVIII-Fc fusion protein has the sequence set forth in SEQ ID NO.
- the present application also provides a nucleic acid encoding a recombinant single-stranded human FVIII-Fc fusion protein of the sequence SEQ ID NO. 1, the sequence of which is set forth in SEQ ID NO.
- the application also provides an expression vector comprising the nucleic acid described above.
- the present application also provides a cell line expressing the aforementioned recombinant single-chain human FVIII-Fc fusion protein.
- the application also provides a composition comprising the aforementioned recombinant single chain human FVIII-Fc fusion protein, and a pharmaceutically acceptable carrier.
- the above composition further comprises a FVIII heavy chain, a FVIII light chain-Fc.
- the present application also provides the use of the above recombinant single-stranded human FVIII-Fc fusion protein for the preparation of a medicament for treating hemophilia A.
- the recombinant single-stranded human FVIII-Fc fusion protein provided herein achieves comparable clotting activity to commercially available recombinant FVIII and has an increased half-life.
- the fusion protein of the present application does not form a homodimer, the yield of the fusion protein is extremely high, and the target protein can be obtained by a conventional protein A purification process, thereby reducing the production cost. .
- Figure 1 shows the cell growth curve
- Figure 2 shows the trends of residual sugar and pH in cell culture fluid.
- Figure 3 is a SEC detection result of the purified recombinant single-stranded human FVIII-Fc fusion protein.
- Figure 4 shows the results of SDS-PAGE detection of the purified recombinant single-stranded human FVIII-Fc fusion protein.
- Figure 5 is a statistical result of the amount of bleeding in each group of animals.
- Figure 6 is a statistical result of bleeding time of each group of animals.
- reagents, vectors, and host cells used in the examples are commercially available.
- the methods employed in the examples are all conventional methods in the art unless otherwise stated.
- SEQ ID NO. 1 is the amino acid sequence of the recombinant single-stranded human FVIII-Fc fusion protein, such that the C-terminus of FVIII is linked to the N-terminus of Fc via the spacer sequence GS, wherein the B-domain deleted human FVIII from position 20 to 1457 Sequence; position 1 to position 19 is the natural signal peptide of human FVIII by which the fusion protein is secreted outside the cell.
- the Fc domain preferentially selects human IgG1, wherein the N297G (EU encoding) mutation removes the ADCC effect; the two CH2-CH3 domains are joined into a single strand by the flexible polypeptide sggsstasgsgsgsggsgtagssggagssggsttaggsasgsgstgsgtggassggasgasg and introduced in the single-stranded Fc fragment near the N-terminal CH3
- the T366S (EU coding), L368A and Y407V mutations introduced a T366W mutation in CH3 near the C-terminus.
- F8-59L The recombinant single-stranded human FVIII-Fc fusion protein is also referred to as F8-59L hereinafter.
- the full-length gene sequence was codon-optimized according to the preference of CHO cells (Chinese hamster ovary cells), and the optimized nucleic acid sequence is shown in SEQ ID NO. 2, which was cloned into the eukaryotic expression vector pcDNA3.1-SD vector by means of NheI and EcoRI. on.
- This vector contains the DHFR gene, which enables high-level expression of single-stranded human FVIII-Fc by co-amplification of DHFR and fusion protein genes.
- CHO DG44 Transfection of host cells (CHO DG44) by electroporation: using a Bio-Rad electrorotator, a 4 mm electric rotor, setting the voltage to 280 V and the shock time to 25 ms. Each shock was 1 ⁇ 10 7 cells, 40 ⁇ g of plasmid, and the total volume was 0.7 ml. After electroporation, the cells were transferred to shake flasks containing 30 ml of growth medium. After 24 hours of culture, the medium was changed to a screening medium containing 200 nM MTX, and seeded in a 96-well plate at 1000 cells/well. The cells were cultured for about 2 weeks until the clonal confluence rate reached 80% or higher.
- the expression level was analyzed by sandwich ELISA using anti-human FVIII antibody and anti-human IgG antibody, and clones F8-P3.1-59 with relatively high expression were screened out.
- -2C2 sequentially transferred into a 24-well plate, a 6-well plate, a T25 cell culture flask, and a cell culture shake flask to expand the culture.
- the concentration of MTX in the medium was increased to 500 nM when transferred to a 24-well plate.
- a recombinant single-stranded human FVIII-Fc fusion protein was produced using the Fed-batch method.
- the cells were seeded at a concentration of 0.8 ⁇ 10 6 /mL into a 5 L bioreactor (Biobundle 5L, Applikon) under the following conditions: 37 ° C, pH 6.95 ⁇ 0.15, dissolved oxygen (DO) 40%, and fed stream after 3 to 4 days of culture. Add culture. On day 10, cell viability dropped to 88%, fermentation was stopped and the supernatant was collected for purification. When the can was placed, the yield in the culture supernatant was estimated to be 272.3 IU/ml based on the FVIII activity measured by the chromogenic substrate method (see Example 2).
- Figure 1 shows the cell growth curve of F8-P3.1-59-2C2.
- the highest cell growth density reached 11.5 ⁇ 10 6 /mL, and the cell doubling time was 37.8 h.
- Figure 2 shows the trends of residual sugar and pH in cell culture fluid.
- the pH is 6.95 ⁇ 0.15.
- the glucose consumption is about 2.5g per day.
- the cell consumes less sugar.
- the amount of sugar consumed gradually increases up to 4g/L.
- the cell culture supernatant was collected by centrifugation and filtered through a 0.22 ⁇ m nitrocellulose filter.
- the purification process is as follows:
- Protein A affinity chromatography for preliminary capture and concentration--G25 displacement buffer--Q-HP anion chromatography to remove impurities such as DNA and HCP, and remove a certain amount of polymer components--Superdex 200 molecular sieve chromatography to remove polymer The sample was replaced into a formulation buffer system.
- the active FVIII isolated and purified from plasma or cell culture supernatant consists of two chains, the light chain and the heavy chain, which are non-covalently linked by a divalent ion (Ca 2+ ).
- the FVIII activity of the fusion protein was determined by using Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086). The detection principle is as follows: When activated by thrombin, FVIIIa binds to FIXa to form an enzyme complex in the presence of phospholipids and calcium ions. The activator factor X is then converted to its active form Xa. Activated Xa in turn cleaves the specific chromogenic substrate (S-2765), releasing the chromophoric group pNA.
- IU/mg biological activity (IU/ml) / protein content (mg / ml)
- the coagulation method for determining the biological activity of FVIII was obtained by correcting the ability of the FVIII factor-deficient plasma to cause prolonged clotting time.
- a kit Coagulation Factor VIII Deficient Plasma (Cat. No. OTXW17) manufactured by the German company Siemens was used.
- the detection method is firstly known as a potency FVIII activity standard, WHO International Standard 8th International Standard Factor VIII Concentrate (Cat. No.
- IU/mg biological activity (IU/ml) / protein content (mg / ml)
- the deletion of the recombinant B domain The specific activity was 9110-13700 IU/mg.
- the molecular weight is 170 kDa, and the theoretical molecular weight of the recombinant single-stranded human FVIII-Fc is 221 kDa.
- the addition of the C-terminal single-chain Fc does not affect the activity of FVIII.
- the hemostatic activity of the fusion protein F8-59L prepared in Example 1 in HemA mice was evaluated using the FVIII gene knockout homozygous HemA mouse tail bleeding model. 8-12 weeks old male HemA mice (purchased from Shanghai Southern Model Bio-Limited Co., Ltd.) were selected. After one week of adaptive feeding, the mice were randomly divided into 6 groups, and a single intravenous injection of different active doses of fusion protein F8 was given. -59L or control drug Renjie (Xyntha, Pfizer), another HemA mouse + vector group (Vehicle) and normal mouse C57BL / 6 + vector group. The grouping of animals is shown in Table 2 below:
- mice Prior to administration, mice were first anesthetized with 1.0% pentobarbital sodium (Sigma) at a dose of 0.1 ml/10 g, and then the mice were placed on a heating pad at 37 ° C to maintain their body temperature.
- the tail of the mouse was immersed in warm water of 37 ° C for 10 minutes to dilate the tail vein, and then administered by tail vein injection according to the above table. After 10 minutes of administration, it was cut at a distance of 1.5 cm from the tail end of the mouse, and the tail was quickly immersed in a centrifuge tube containing about 13 ml of preheated physiological saline, and the timing was started. If the bleeding stops within 30 minutes, record the bleeding time and the amount of bleeding.
- the bleeding time exceeds 30 minutes, it is recorded as 30 minutes.
- the amount of bleeding (ml) (weight of centrifuge tube after blood collection (g) - weight of centrifuge tube (g) before blood collection) / 1.05.
- the rat tail was taken out from the saline tube. The rebleeding was recorded every 10 minutes for 24 hours and the number of mice survived was recorded. All data in mean ⁇ standard error T-test analysis was used for comparison between the experimental groups. The analysis software used Graphpad Prism 5.0, p ⁇ 0.05 was considered statistically significant.
- the F8-59L 90IU/kg group showed a significant reduction in bleeding volume and a significantly shorter bleeding time (p ⁇ 0.05; p ⁇ 0.05), and the F8-59L 270 IU/kg group significantly reduced the amount of bleeding.
- the bleeding time was significantly shortened (p ⁇ 0.01; p ⁇ 0.01).
- the amount of bleeding in the F8-59L 270 IU/kg group was reduced (p ⁇ 0.05), indicating that the fusion protein F8-59L pair
- the hemostatic effect of acute hemorrhage in HemA mice has a dose-response relationship (see Table 3 for detailed results).
- bleeding time is more than 30 minutes, recorded as 1800 seconds
Abstract
提供了一种重组单链人FVIII-Fc融合蛋白,其中,FVIII是B结构域缺失的人FVIII;Fc来自包含N297G突变的人IgG1,两个CH2-CH3结构域通过柔性多肽连接成一条单链,在靠近N端的CH3中引入T366S、L368A和Y407V突变,在靠近C端的CH3中引入T366W突变。由于在Fc链内引入了点突变,本申请的重组单链人FVIII-Fc融合蛋白不会形成同源二聚体。
Description
本申请要求于2016年12月23日提交中国专利局、申请号为201611203557.0发明名称为“重组单链人FVIII-Fc融合蛋白及其应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本申请涉及重组FVIII-Fc融合蛋白领域,更具体地涉及B结构域缺失的人FVIII与特定的单链Fc连接形成的融合蛋白。
A型血友病是由凝血因子VIII(FVIII)活性的缺乏或功能障碍造成的一种遗传性出血障碍。补充有活性的FVIII是治疗A型血友病的有效措施。内源的FVIII的循环半衰期是12-14小时,因而每周需要进行几次预防性治疗,为了提高患者的依从性,减少FVIII每周施用的次数,研究者一直致力于开发半衰期延长的FVIII。
利用活性蛋白与IgG的Fc结构域连接构成融合蛋白来延长活性蛋白在体内的半衰期,是目前本领域技术人员常采用的手段之一。但是早期的Fc融合蛋白均是将两个活性蛋白与一个Fc片段融合形成二价分子。FVIII分子量较大,是目前重组技术表达的分子量最大的活性蛋白,并且其需要与磷脂、FIXa和FX形成复合物才能发挥凝血活性,因此,二价分子形式的FVIII-Fc蛋白会由于空间位阻效应而导致FVIII活性降低或丧失。
专利CN201080062950.7公开了一种FVIII-Fc嵌合蛋白,其中该嵌合蛋白包含两条链,一条链包括FVIII与IgG1的铰链区-CH2-CH3,另一条链仅包括IgG1的铰链区-CH2-CH3,二条链通过Fc区二聚化而形成杂合蛋白。但是,在该嵌合蛋白生产过程中,由于可同时产生“铰链区-CH2-CH3”同源二聚形式的分子,因此嵌合FVIII-Fc的产量极低,而且不能通过常规的protein A纯化工艺获得目的蛋白,只能采用VIIIselect(GE Healthcare)来纯化,从而极大的增加了生产成本。
发明内容
本申请目的在于提供一种新的重组单链人FVIII-Fc融合蛋白,其中Fc片段中的两个CH2-CH3结构域通过柔性多肽连接成一条单链。并采用真核细胞表达系统进行融合蛋白表达。所表达的重组单链人FVIII-Fc可用常规protein A纯化工艺获得较高纯度蛋白。
本申请的技术方案如下:
本申请首先提供了一种重组单链人FVIII-Fc融合蛋白,其中,FVIII是B结构域缺失的人FVIII;Fc来自包含N297G突变的人IgG1,两个CH2-CH3结构域通过柔性多肽连接成一条单链,在靠近N端的CH3中引入T366S、L368A和Y407V突变,在靠近C端的CH3中引入T366W突变。
优选地,所述FVIII与Fc直接连接或通过柔性多肽连接。
优选地,所述柔性多肽由至少一种选自G、S、A、T的氨基酸组成。
优选地,重组单链人FVIII-Fc融合蛋白,其序列如SEQ ID NO.1所示。
本申请还提供了编码序列如SEQ ID NO.1所示的重组单链人FVIII-Fc融合蛋白的核酸,其序列如SEQ ID NO.2所示。
本申请还提供了包含上述的核酸的表达载体。
本申请还提供了表达前述的重组单链人FVIII-Fc融合蛋白的细胞株。
本申请还提供了一种组合物,其包含前述的重组单链人FVIII-Fc融合蛋白,和药学上可接受的载体。
优选地,上述组合物其还包括FVIII重链、FVIII轻链-Fc。
本申请还提供了上述的重组单链人FVIII-Fc融合蛋白在制备治疗A型血友病的药物中的用途。
本申请提供的重组单链人FVIII-Fc融合蛋白达到了与市售的重组FVIII相当的凝血活性,而且半衰期延长。另外,由于在Fc链内引入了点突变,本申请的融合蛋白不会形成同源二聚体,融合蛋白的产量极高,而且能够通过常规的protein A纯化工艺获得目的蛋白,降低了生产成本。
为了更清楚地说明本申请实施例和现有技术的技术方案,下面对实施例和现有技术中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为细胞生长曲线。
图2为细胞培养液残糖和PH变化趋势。
图3为纯化后的重组单链人FVIII-Fc融合蛋白的SEC检测结果。
图4为纯化后的重组单链人FVIII-Fc融合蛋白的SDS-PAGE检测结果。
图5为各组动物的出血量的统计结果。
图6为各组动物的出血时间的统计结果。
为使本申请的目的、技术方案、及优点更加清楚明白,以下参照附图并举实施例,对本申请进一步详细说明。显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
实施例所用到的试剂、载体、宿主细胞均市售可得。如无特殊说明,实施例所采用的方法均为本领域的常规方法。
实施例1制备重组单链人FVIII-Fc融合蛋白
1)表达载体的构建
SEQ ID NO.1为重组单链人FVIII-Fc融合蛋白的氨基酸序列,使FVIII的C端通过间隔序列GS与Fc的N端连接,其中,20位到1457位为B结构域缺失的人FVIII序列;1位到19位为人FVIII的天然信号肽,融合蛋白借助于该信号肽分泌到细胞外。Fc结构域优先选择人IgG1,其中N297G(EU编码)突变以去除ADCC效应;两个CH2-CH3结构域通过柔性多肽 sggsstasgsgsggsgtagssggagssggsttaggsasgsgstgsgtggassggasgasg连接成一条单链,并在单链Fc片段中靠近N端的CH3中引入T366S(EU编码)、L368A和Y407V突变,在靠近C端的CH3中引入T366W突变。这种设计是为了更好地形成链内Fc结构,而避免形成“FVIII-Fc-Fc”同源二聚体。在后文中也将重组单链人FVIII-Fc融合蛋白称为F8-59L。
全长基因序列根据CHO细胞(中国仓鼠卵巢细胞)偏爱进行密码子优化,优化的核酸序列如SEQ ID NO.2所示,其借助于NheI和EcoRI克隆到真核表达载体pcDNA3.1-SD载体上。该载体包含DHFR基因,可通过DHFR和融合蛋白基因的共扩增来实现单链人FVIII-Fc的高水平表达。
2)高表达细胞株的筛选
采用电穿孔的方法转染宿主细胞(CHO DG44):使用Bio-Rad电转仪,4mm电转杯,设置电压为280V和电击时间为25毫秒。每次电击1×10
7个细胞,质粒40μg,总体积为0.7ml。电转后细胞转入含有30ml生长培养基的摇瓶中培养。培养24小时后,将培养基换成含200nM MTX的筛选培养基中,并以1000个细胞/孔接种于96孔板中。细胞培养2周左右,直到克隆汇合率达到80%或以上,用抗人FVIII抗体和抗人IgG抗体,采用夹心ELISA方法分析表达量,筛选出表达量相对高的克隆F8-P3.1-59-2C2,依次转入24孔板、6孔板、T25细胞培养瓶和细胞培养摇瓶中扩大培养。在转入24孔板时,将培养基中MTX的浓度提高到500nM。
3)融合蛋白的生产
采用Fed-batch的方法生产重组单链人FVIII-Fc融合蛋白。细胞以0.8×10
6个/mL接种到5L生物反应器(Biobundle 5L,Applikon),反应条件为:37℃、pH 6.95±0.15、溶氧(DO)40%,培养3到4天后补料流加培养。第10天时,细胞存活率降到88%,终止发酵并收集上清液进行纯化。下罐时,根据发色底物法(见实施例2)测得的FVIII活性估算培养上清中的产量为272.3IU/ml。
图1为F8-P3.1-59-2C2细胞生长曲线。细胞生长最高密度达到11.5×10
6个/mL,细胞倍增时间为37.8h。
图2为细胞培养液残糖和PH变化趋势。在整个发酵过程中,pH全程6.95±0.15;在第3-5天,每天葡萄糖消耗约2.5g,第五天,培养温度降低后,细胞耗糖减少,在第7-9天时,随着蛋白进入高速表达期,耗糖量逐渐增加,最多可达4g/L。
4)融合蛋白的纯化
离心收集细胞培养上清,用0.22μm的硝酸纤维素过滤器过滤。其纯化流程如下:
Protein A亲和层析进行初步捕获及浓缩--G25置换缓冲液--Q-HP阴离子层析去除DNA及HCP等杂质,并去除一定量的聚体成分--Superdex 200分子筛层析去除聚体并将样品置换至制剂缓冲体系中。
从血浆或细胞培养上清中分离纯化得到的活性FVIII由两条链组成--轻链和重链,二者之间通过二价离子(Ca
2+)非共价连接。
纯化后的重组单链人FVIII-Fc融合蛋白的SEC检测结果见图3,蛋白纯度为97.80%。SDS-PAGE检测结果见图4,和预测一致,非还原和还原电泳都包括三条带,分别为FVIII重链(约90KDa),FVIII轻链-Fc(约130kDa)和全长重组人FVIII-Fc(约220kDa)。
实施例2重组融合蛋白的生物活性分析
1.发色底物法测定融合蛋白的生物学活性
采用Chromogenix Coatest SP FVIII试剂盒(Chromogenix,Ref.K824086)测定融合蛋白的FVIII活性,其检测原理如下:当被凝血酶激活后,FVIIIa在磷脂和钙离子存在下,与FIXa结合形成酶复合物,继而可激活因子X转变成其活性形式Xa。活化的Xa继而可使特异性发色底物(S-2765)发生裂解,释放发色基团pNA。在405nm下测定所产生pNA的量,即可知与其量直接成正比关系的FXa的活性大小,其中在体系中因子IXa和因子X的含量是一定且过量的,FXa的活性仅与FVIIIa的含量多少直接相关。用发色法测定纯化的重组单链人FVIII-Fc的生物学活性为899.4IU/ml。根据以下公式计算其比活性为9993.3IU/mg。
比活性(IU/mg)=生物学活性(IU/ml)/蛋白含量(mg/ml)
2.一期法(凝固法)测定融合蛋白的生物学活性
凝固法测定FVIII生物学活性是通过纠正FVIII因子缺失血浆所导致凝固时间延长的能力而获得的。采用德国Siemens公司生产的试剂盒Coagulation Factor VIII Deficient Plasma(Cat.No.OTXW17)。检测方法首先是已知效价的FVIII活性标准品WHO International Standard 8th International Standard Factor VIII Concentrate(Cat.No.07/350)用5%乏FVIII血浆稀释至1IU/ml,再分别进行稀释10倍、20倍、40倍、80倍,并与乏FVIII基质血浆混合,测定部分凝血活酶时间(APTT),以FVIII活性标准品溶液效价(IU/ml)的对数对应其相应的凝固时间(s)的对数作直线回归,建立标准曲线。再将待测样本经适度稀释后与乏FVIII基质血浆混合,进行APTT测定。通过代入标准曲线,可知待测样品FVIII的效价为多少,据此可求算出待测样品FVIII的比活性大小,单位为IU/mg。用凝血法直接测定纯化的重组单链人FVIII-Fc的生物学活性为725.6IU/ml。根据以下公式计算其比活性为10321.1IU/mg。
比活性(IU/mg)=生物学活性(IU/ml)/蛋白含量(mg/ml)
根据报道,重组B结构域缺失的
的比活性为9110-13700IU/mg。
的分子量为170kDa,而重组单链人FVIII-Fc的理论分子量为221kDa,考虑到两个蛋白分子量的差异,说明C末端单链Fc的加入不影响FVIII的活性。
实施例3重组融合蛋白对血友病A(HemA)小鼠急性出血的止血作用
用FVIII基因剔除纯合子HemA小鼠断尾出血模型(tail clip bleeding model)评估实施例1所制备的融合蛋白F8-59L在HemA小鼠体内的止血活性。选取8-12周龄雄性HemA小鼠(购自上海南方模式生物责任有限公司),适应性饲养一周后将小鼠随机分为6组,分别单次尾静脉注射给予不同活性剂量的融合蛋白F8-59L或对照药物任捷(Xyntha,辉瑞),另设HemA小鼠+载体组(Vehicle)和正常小鼠C57BL/6+载体组。动物分组情况见下表2:
表2:融合蛋白F8-59L对HemA小鼠止血效果实验动物分组情况
给药前,首先以1.0%戊巴比妥钠(Sigma公司)按照0.1ml/10g剂量腹腔注射麻醉小鼠,然后将小鼠置于37℃的加热垫上以保持其体温。将小鼠尾巴浸入37℃的温水中10分钟,使尾静脉扩张,然后按照上表方式尾静脉注射给药。给药10分钟后,在距小鼠尾末端1.5cm处剪断,迅速将尾端浸入装有约13ml预热生理盐水的离心管中,并开始计时。如果出血在30分钟内停止,记录出血时间和出血量。如果出血时间超过30分钟,则记为30分钟。出血量(ml)=(采血后离心管重量(g)-采血前离心管重量(g))/1.05。30分钟后,将鼠尾从生盐水管中取出。在24小时内,每隔10分钟观察记录复出血情况,并记录小鼠存活数量。所有数据以均数±标准误
表示,各实验组间比较采用t-test检验分析,分析软件采用Graphpad Prism 5.0,p<0.05认为有统计学意义。
从图5和图6中各组动物的出血量和出血时间的统计结果分析,HemA小鼠给予F8-59L 270IU/kg 10分钟后,其出血时间和出血量与C57+载体组接近;小鼠给药F8-59L 90IU/kg后,在出血时间和出血量上与C57+载体组差异不显著;与HA+载体组比较,F8-59L 30IU/kg给药组的出血量显著减少、出血时间显著缩短(p<0.01;p<0.01),说明F8-59L促凝血效果明显,可以作为 血友病等凝血因子缺乏症发生急性出血情况的有效凝血剂。与F8-59L 30IU/kg给药组比较,F8-59L 90IU/kg组出血量显著减少、出血时间显著缩短(p<0.05;p<0.05),F8-59L 270IU/kg组的出血量显著减少、出血时间显著缩短(p<0.01;p<0.01);另外与F8-59L 90IU/kg组比较,F8-59L 270IU/kg组的出血量减少(p<0.05),说明融合蛋白F8-59L对HemA小鼠急性出血的止血作用具有一定的剂量-效应关系(详细结果见表3)。
表3:各组HemA小鼠断尾试验的出血时间、出血量和存活率统计
注:a,出血时间超过30分钟,记做1800秒
以上所述仅为本申请的较佳实施例而已,并不用以限制本申请,凡在本申请的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本申请保护的范围之内。
Claims (10)
- 一种重组单链人FVIII-Fc融合蛋白,其中,FVIII是B结构域缺失的人FVIII;Fc来自包含N297G突变的人IgG1,两个CH2-CH3结构域通过柔性多肽连接成一条单链,在靠近N端的CH3中引入T366S、L368A和Y407V突变,在靠近C端的CH3中引入T366W突变。
- 根据权利要求1所述的重组单链人FVIII-Fc融合蛋白,所述FVIII与Fc直接连接或通过柔性多肽连接。
- 根据权利要求1或2所述的重组单链人FVIII-Fc融合蛋白,所述柔性多肽由至少一种选自G、S、A、T的氨基酸组成。
- 根据权利要求3所述的重组单链人FVIII-Fc融合蛋白,其序列如SEQ ID NO.1所示。
- 编码权利要求4所述的重组单链人FVIII-Fc融合蛋白的核酸,其序列如SEQ ID NO.2所示。
- 包含权利要求5所述的核酸的表达载体。
- 表达权利要求1至4中任一项所述的重组单链人FVIII-Fc融合蛋白的细胞株。
- 一种组合物,其包含权利要求1至4中任一项所述的重组单链人FVIII-Fc融合蛋白,和药学上可接受的载体。
- 根据权利要求8所述的组合物,其还包含FVIII重链、FVIII轻链-Fc。
- 权利要求1至4中任一项所述的重组单链人FVIII-Fc融合蛋白在制备治疗A型血友病的药物中的用途。
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